1. Field of the Invention
The present invention relates to a method for the culture of parasites of the Babesia canis species. It also relates to the application of this method to the preparation of antigenes and vaccines against canine babesioses, as well as to the antigenes and vaccines obtained.
2. Description of the Prior Art
The preparation of vaccines against babesiosis has for a long time posed practically insurmountable problems. These difficulties were linked to the impossibility of breeding parasites in vitro on an industrial scale as well as to the difficulties of detecting and isolating useful antigenes, and more especially protective antigenes.
A process for breeding Babesia was proposed in Erp. et al. Am. Jour Trop. Med. Hva., 27 (5): pp. 1061-1064 (1978). This method consisted in propagating parasites in a culture of erythrocytes with mechanical stirring and with an increased carbonic gas content. This process did not however allow mass production to be reached.
The attempts made for producing babesiosis parasites using techniques close to the Plasmodium culture, described in Trager, et al., Science, 193: pp. 673-675 (1976) or Speer et al., Z. Parasitenk, 50: pp. 237-244 (1976), have not been successful.
More recently, a method has been proposed by Miodrag Ristic and Michael G. Levy, in the European patent No. 18580 for large scale production of these parasites. This method is based on incubation in erythrocytic cells in a culture medium suitable for the erythrocytes, to which is added 30 to 50% of serum, this incubation taking place statically in the presence of a controlled atmosphere containing 3 to 6% of carbonic gas and under conditions allowing the hemoglobin of the erythrocytes in the deoxidized state, to be kept at a temperature of the order of 35.degree. to 38.degree. C. This method has proved efficient for the large scale production of Babesia bovis, but not of Babesia canis.
It is apparent that it would be interesting to able to produce Babesia canis on a large scale under practical and economic conditions so as to be able to manufacture a vaccine against canine piroplasmosis.
The invention provides then such a method which allows the Babesia canis parasite to be produced on a large scale simply and inexpensively.
Another object of the invention is to provide such a method for preparing antigenes or vaccines from the supernatant culture products.
Another object of the invention is to provide a method for applying the culture process of the invention to the preparation of antigenes or vaccines.
A further object of the invention is to provide antigenes, purified or not, and vaccines, purified or not, from floating culture products.
The invention provides then a method for the culture of parasites of the Babesia canis species, in which erythrocytes infected by the parasite are incubated in a culture medium suitable for the erythrocytes, in the presence of a homologous serum, in the substantially static state, characterized in that the culture is placed in a normal atmosphere to incubate.
By normal atmosphere in the sense of the present invention is meant an atmosphere composed essentially of oxygen and nitrogen and devoid of any considerable carbonic gas content.
In a particularly advantageous embodiment of the invention, the culture is placed first of all to incubate in a high temperature, more particularly between 34.degree. and 38.degree. C., then at a low temperature, especially between 0.degree. and 10.degree. C.
Preferably, the incubation at high temperature lasts for about eight hours and at low temperature about sixteen hours. However, the incubation time at high temperature may be increased and the duration reduced during which the culture is at low temperature, this latter duration not however being preferably less than six to eight hours.
At the end of the low temperature incubation the supernatant medium is decanted and collected and is replaced by new medium, after which the culture is again placed in a high temperature to incubate, then at a lower temperature and from time to time the hematocrit is adjusted.
Advantageously, the erythrocyte concentration, i.e. the hematocrit, is of the order of 8 to 12% and preferably 10%.
Also advantageously, the culture medium comprises between 20% and 3% and preferably 5% of dog serum.
The preferred culture medium is Ham's medium F10 or F12 containing a little glucose and suitably buffered. Thus the medium may for example comprise glucose at 1g/l a Hepes buffer and bicarbonate at about 1.2%.
However other usual media may be used, such for example as 199 with Hanks salts or RPMI 1640. The culture media must be compatible with the survival of the erythrocytes and must contain the elements for breeding the parasites.
This may be obtained by starting with rich culture media and making suitable tests of them.
In the case where the culture method is intended to produce substances for administration to animals, for example a vaccine, the culture is advantageously carried out in the presence of an antibiotic such for example as gentamycine.
The culture is carried out at a suitable pH for developing the parasites, this pH being of the order of 7.3 to 7.5 and preferably of the order of 7.4.
In accordance with the invention, the suoernatant culture product is collected at the end of the low temperature incubation period. This supernatant product may be used either directly, or after purification steps, for forming antigenes and/or a vaccine against canine pyroplasmosis.
Preferably, the supernatant product is collected when the parasite concentration reaches 10.sup.8 parasites/ml.
Advantageously, the supernatant product is centrifugated so as to eliminate the cells, the parasites and the cell remains and parasite remains, after which sterilizing filtration is carried out. The supernatant product may then be advantageously concentrated, for example by ultra-filtration.
For preparing a vaccine, the possible contaminants of the supernatant product are then advantageously inactivated for example with formol or betapropiolactone or ethyl-ethylenimine.
The vaccine is preferably in liophilised form.
The vaccine may be administered subcutaneously.
A vaccine against piroplasmosis, in accordance with the invention, is characterized in that it contains soluble and concentrated Babesia canis antigenes coming from a culture supernatant of Babesia canis on erythrocytes. cytes.
Preferably, the dose contains at most 0.120 mg of formol and 20% of betapropiolactone or ethyl-ethylenimine.
The solvent of the liophilized vaccine comprises preferably an adjuvant and is for example in the form of a solution of saponin, for example at 0.5 mg/ml.